Group Title: Arthritis Research & Therapy
Title: High prevalence of autoantibodies to RNA helicase A in Mexican patients with systemic lupus erythematosus
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 Material Information
Title: High prevalence of autoantibodies to RNA helicase A in Mexican patients with systemic lupus erythematosus
Physical Description: Book
Language: English
Creator: Va¡zquez-Del Mercado, Monica
Palafox-Sanchez, Claudia
Munoz-Valle, Jose
Orozco-Barocio, Gerardo
Oregon-Romero, Edith
Navarro-Hernandez, Rosa
Salazar-Paramo, Mario
Armendariz-Borunda,J uan
Gamez-Nava, Jorge
Gonzalez-Lopez, Laura
Chan, Jason
Chan, Edward
Satoh, Minoru
Publisher: Arthritis Research and Therapy
Publication Date: 2010
 Notes
Abstract: INTRODUCTION:Autoantibodies to RNA helicase A (RHA) were reported as a new serological marker of systemic lupus erythematosus (SLE) associated with early stage of the disease. Anti-RHA and other autoantibodies in Mexican SLE patients and their correlation with clinical and immunological features were examined.METHODS:Autoantibodies in sera from 62 Mexican SLE patients were tested by immunoprecipitation of 35S-labeled K562 cell extract and enzyme-linked immunosorbent assay (anti-U1RNP/Sm, ribosomal P, ß2GPI, and dsDNA). Anti-RHA was screened based on the immunoprecipitation of the 140-kDa protein, the identity of which was verified by Western blot using rabbit anti-RHA serum. Clinical and immunological characteristics of anti-RHA-positive patients were analyzed.RESULTS:Anti-RHA was detected in 23% (14/62) of patients, a prevalence higher than that of anti-Sm (13%, 8/62). Prevalence and levels of various autoantibodies were not clearly different between anti-RHA (+) vs. (-) cases, although there was a trend of higher levels of anti-RHA antibodies in patients without anti-U1RNP/Sm (P = 0.07). Both anti-RHA and -Sm were common in cases within one year of diagnosis; however, the prevalence and levels of anti-RHA in patients years after diagnosis did not reduce dramatically, unlike a previous report in American patients. This suggests that the high prevalence of anti-RHA in Mexican patients may be due to relatively stable production of anti-RHA.CONCLUSIONS:Anti-RHA was detected at high prevalence in Mexican SLE patients. Detection of anti-RHA in races in which anti-Sm is not common should be clinically useful. Racial difference in the clinical significance of anti-RHA should be clarified in future studies.
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General Note: M3: 10.1186/ar2905
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Bibliographic ID: UF00099893
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: Open Access: http://www.biomedcentral.com/info/about/openaccess/
Resource Identifier: issn - 1478-6354
http://arthritis-research.com/content/12/1/R6

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Vazquez-Del Mercado et al. Arthritis Research & Therapy 2010, 12:R6
http://arthritis-research.com/content/12/1/R6


High prevalence of autoantibodies to RNA

helicase A in Mexican patients with systemic

lupus erythematosus

Monica V6zquez-Del Mercado '2, Claudia A Palafox-Sanchezl, Jose F Munoz-Vallel, Gerardo Orozco-Barocio3,
Edith Oregon-Romero', Rosa E Navarro-Hernandez', Mario Salazar-Paramo4, Juan Armendariz-Borunda5,
Jorge I Gamez-Nava4, Laura Gonzalez-Lopez6, Jason YF Chan7, Edward KL Chan8, Minoru Satoh7'9*


Abstract
Introduction: Autoantibodies to RNA helicase A (RHA) were reported as a new serological marker of systemic
lupus erythematosus (SLE) associated with early stage of the disease. Anti-RHA and other autoantibodies in
Mexican SLE patients and their correlation with clinical and immunological features were examined.
Methods: Autoantibodies in sera from 62 Mexican SLE patients were tested by immunoprecipitation of 35 -labeled
K562 cell extract and enzyme-linked immunosorbent assay (anti-UIRNP/Sm, ribosomal P, p2GPI, and dsDNA). Anti-
RHA was screened based on the immunoprecipitation of the 140-kDa protein, the identity of which was verified by
Western blot using rabbit anti-RHA serum. Clinical and immunological characteristics of anti-RHA-positive patients
were analyzed.
Results: Anti-RHA was detected in 23% (14/62) of patients, a prevalence higher than that of anti-Sm (13%, 8/62).
Prevalence and levels of various autoantibodies were not clearly different between anti-RHA (+) vs. (-) cases,
although there was a trend of higher levels of anti-RHA antibodies in patients without anti-UIRNP/Sm (P = 0.07).
Both anti-RHA and -Sm were common in cases within one year of diagnosis; however, the prevalence and levels of
anti-RHA in patients years after diagnosis did not reduce dramatically, unlike a previous report in American
patients. This suggests that the high prevalence of anti-RHA in Mexican patients may be due to relatively stable
production of anti-RHA.
Conclusions: Anti-RHA was detected at high prevalence in Mexican SLE patients. Detection of anti-RHA in races in
which anti-Sm is not common should be clinically useful. Racial difference in the clinical significance of anti-RHA
should be clarified in future studies.


Introduction
Systemic autoimmune rheumatic diseases such as sys-
temic lupus erythematosus (SLE), scleroderma (systemic
sclerosis), and polymyositis/dermatomyositis are serolo-
gically characterized by the production of autoantibodies
to cellular constituents [1,2]. Although autoantibodies
target various proteins, protein complexes, protein-
nucleic acid complexes, and nucleic acids, selection of
the target antigens is not a random event; rather, there

* Correspondence' Minoru Satoh@medicineufledu
'Division of Rheumatology and Clinical Immunology, Department of
Medicine, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610-
0424, USA


can be a tight link between the specificity of autoantibo-
dies each patient produces and the diagnosis or certain
clinical symptoms. Some of the specificities are detected
almost exclusively in patients with certain clinical diag-
nosis and considered pathognomonic. Anti-Sm and dou-
ble-stranded DNA (dsDNA) antibodies are highly
specific for the diagnosis of SLE and are included in the
classification criteria [3]. While anti-dsDNA antibodies
are found in approximately 70% of patients with SLE,
their production fluctuates depending on the lupus
activity and treatment they receive. Production of anti-
Sm antibodies is generally considered more stable and is
found in approximately 15% of patients with SLE;


0 2010 Vazques-Del Mercado et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Biole led Central Creative Commons Attribution License (http//creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.


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Vazquez-Del Mercado et al. Arthritis Research & Therapy 2010, 12:R6
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however, it is common in African-Americans and is low
in prevalence in Caucasians [4]. Anti-ribosomal P and
anti-PCNA (proliferating cell nuclear antigen) antibodies
found in approximately 10% and approximately 2% of
patients with SLE also are considered specific for SLE
[1]. We have recently reported that, in addition to these
classic markers, autoantibodies to RNA helicase A
(RHA, also known as DNA helicase II), a 3'-5' dsDNA/
RNA helicase [5] that belongs to the DExH superfamily
of helicases, are a new serological marker of SLE [1,4].
In the previous report, the rates of prevalence of anti-
RHA were 6% (8/133) in Caucasians, 2.9% (3/103) in
African-Americans, and 12% (3/25) in the Latin popula-
tion in the US. Another earlier report was also from the
US [6]. Except for preliminary data suggesting that
approximately 10% of Japanese patients with SLE are
also positive [7], anti-RHA in other countries has not
been reported. Anti-RHA is also unique in that it is
associated with the early stage of the disease, typically
within a year of diagnosis of SLE. However, the number
in the Latin population was too small to analyze in the
previous study [4]. In the present study, we determined
the prevalence of anti-RHA and examined the clinical
and immunological characteristics of anti-RHA-positive
Mexican patients with SLE.

Materials and methods
Patients
Sixty-two consecutive patients with SLE from the
Department of Rheumatology, Hospital General de
Occidente, Zapopan, Jalisco, Mexico, were studied. All
patients fulfilled the 1982 American College of Rheuma-
tology (ACR) SLE classification criteria [3]. Mex-SLE-
DAI (Mexican Systemic Lupus Erythematosus Disease
Activity Index) and Systemic Lupus International Colla-
borating Clinics/ACR Damage Indexes at the beginning
of the study were evaluated [8,9]. Complete blood
count, including lymphocyte count and serum rheuma-
toid factor (CELL-DYN 3500R; Abbott Diagnostics, Chi-
cago, IL, USA), was determined in all subjects.
Information on treatment of the day of sampling,
including use of immunosuppressive drugs (azathiopr-
ine, methotrexate, and cyclophosphamide), chloroquine,
and dose of steroid (milligrams of prednisone per day),
was recorded. The protocol was approved by the institu-
tional review board. This study meets and is in compli-
ance with all ethical standards in medicine, and written
informed consent was obtained from all patients accord-
ing to the Declaration of Helsinki.

Screening of autoantibodies in human sera by
immunoprecipitation
Immunoprecipitation (IP) using 35S-methionine-labeled
K562 cell extract to determine IgG class autoantibodies


was performed using 8 pL of sera as described [10]. Spe-
cificities such as anti-UlRNP, Sm, ribosomal P, Ro, La,
Ku, argonaute 2 (Ago2)/Su, and RNA polymerase II
(RNAP II) were verified using previously described
reference sera. Positive anti-UlRNP was defined based
on the presence of the set of U1RNP proteins (A, B'/B,
C, D1/D2/D3, E/F, and G). Since autoantibodies to
U5RNP without anti-Sm are very rare [11], IP of the
characteristic U5RNP 200-kDa proteins was used to
define anti-Sm (which immunoprecipitates U2, U4-6,
and U5 in addition to U1RNP) [10].
Anti-RHA was first screened based on IP of the 140-
kDa protein. Selected sera were then re-run on 8% SDS-
PAGE to verify that the mobility of the 140-kDa protein
was the same as that of RHA immunoprecipitated by the
reference sera. The identity of the 140-kDa protein as
RHA was further confirmed by IP-Western blot (WB) as
previously described [12]. Briefly, non-radiolabeled K562
cell extract from 5 x 106 cells was immunoprecipitated
with 2 ptL of serum that immunoprecipitated the 140-
kDa protein in 35S-IP. Samples were run on 8% SDS-
PAGE and transferred to a nitrocellulose filter. The filter
was probed by rabbit anti-RHA antiserum (1:2,000, a gift
from Jun-Qi Yang and Michael B Mathews, University of
Medicine and Dentistry of New Jersey, Newark, NJ, USA)
[13] followed by 1:2,000 horseradish peroxidase-labeled
goat IgG F(ab')2 anti-rabbit IgG (y-chain- and light-
chain-specific; SouthernBiotech, Birmingham, AL, USA)
and developed using SuperSignal West Pico Chemilumi-
nescent Substrate (Pierce, Rockford, IL, USA).

Quantification of anti-RHA antibody levels
Levels of anti-RHA were estimated and analyzed using
the Storm Phosphorimager and images were obtained on
a storage phosphor screen (Amersham Biosciences, now
part of GE Healthcare, Little Chalfont, Buckinghamshire,
UK) from 8% SDS-PAGE gels. The integrated density
(that is, the sum of the values of the pixels in the image
or selection; this is equivalent to the product of the area
and the mean gray value) [14] of the RHA on a phos-
phorimage was calculated using ImageJ software
(National Institutes of Health, Bethesda, MD, USA).

Enzyme-linked immunosorbent assay
Sera were tested for IgG anti-UlRNP/Sm, ribosomal
P (P peptide), dsDNA, and P32 glycoprotein I (P32GPI)
(a gift from Junichi Kaburaki, Tokyo Electric Power Com-
pany Hospital, Tokyo, Japan) antibodies by enzyme-linked
immunosorbent assay (ELISA) as described [15,16]. P pep-
tide was COOH-terminal 22 amino acids of human PO
protein [17]. dsDNA was purified using S1 nuclease as
described [15]. Anti-UlRNP/Sm antigen-capture ELISA
was performed as described [16]. Briefly, microtiter plates
(Immobilizer Amino"; Nalge Nunc, Rochester, NY, USA)


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were coated with 3 pg/mL mouse monoclonal antibodies
(mAbs) 2.73 (IgG2a, anti-Ul-70k) [10]. The left half of the
plate was incubated with K562 cell lysate (50 pL/well,
4 x 107/mL), and the right half was incubated with the
blocking buffer as control. After the plate was washed, an
identical set of samples and serially diluted standard
serum (1:500 to serial 1:5 dilutions) were added to the left
and right halves (control for reactivity against mouse IgG)
of the plate. Serum samples were tested at 1:500 and
1:2,500 dilutions, and data from the latter were used for
the analysis. Plates were washed with Tris-buffered saline/
Tween20, incubated with alkaline phosphatase-conjugated
mouse mAbs to human IgG (Sigma-Aldrich, St. Louis,
MO, USA) (1:1,000 dilution), and developed. The optical
density (OD) of 405 nm of wells was converted into units
based on the standard curve, and the units of the corre-
sponding right half (without U1RNP/Sm antigens) were
subtracted from the left half (with antigens) using SoftMax
Pro 4.3 software (Molecular Devices Corporation, Sunny-
vale, CA, USA) [15]. For the detection of anti-P peptide,
dsDNA, and f32GPI, microtiter plates were incubated with
1 to 3 pg/mL of the appropriate antigens, and ELISA was
performed as described previously using 1:500 diluted
sera. ODs were converted into units as described using the
appropriate standard [15].

Statistical analysis
All statistical analysis was performed using Prism 5.0 for
Macintosh (GraphPad Software, Inc., San Diego, CA,
USA). The Fisher exact test and the Mann-Whitney test
or Student t test were used to analyze prevalence and
levels, respectively, of autoantibodies and other data.

Results
A previous study suggested that anti-RHA was asso-
ciated with an early stage of SLE within a few years of
diagnosis [4]. Consistent with this observation, the levels
of anti-RHA dramatically decreased over time. Studies
in mouse models suggested that the environment for
the production of anti-RHA was distinct from that of
anti-small nuclear ribonucleoproteins (anti-snRNPs) or
anti-Su (Ago2) [12,18]. Opposite kinetics of production
of anti-RHA versus anti-snRNPs or anti-ribosomal P,
observed in some human cases, may be reminiscent of
these observations [4,7]. Thus, we examined whether
these findings in the previous human study performed
in an American (mainly Caucasian) population [4] also
apply to Mexican patients with SLE and whether anti-
RHA and anti-snRNPs have a negative correlation.

Screening of anti-RHA antibodies
Anti-RHA antibodies were screened based on the IP of
the 140-kDa protein using 35S-methionine-labeled K562
cell extract as described [4]. Sera selected were run


along with the prototype sera to verify the identical
mobility (Figure la). Several sera with strong reactivities
showing typical degradation patterns (white arrowheads,
see lanes RHA, 4, 5, 7, and 10) were clearly anti-RHA;
however, it is possible that there are other proteins that
co-migrate with RHA. Thus, IP-WB using rabbit anti-
RHA serum was also performed to verify the identity of
proteins immunoprecipitated by each serum (Figure ib).
All 14 sera initially selected by IP were positive by IP-
WB, confirming that the sera indeed had anti-RHA.
Clinical and immunological characteristics of these
14 patients were compared with those of anti-RHA-
negative patients.

Prevalence of anti-RHA antibodies and stage of the
disease
Anti-RHA was found in 23% (14/62) in this cohort of
Mexican patients, a prevalence that was greater than
that of anti-Sm (13%) (Table 1) and much higher than
that of anti-RHA (6%) reported in American patients
[4], whereas the prevalence of other specificities did not
seem to be different from other reports in SLE.
The production of anti-RHA was associated with an
early stage of SLE in American patients [4]. To examine
whether this applies to Mexican patients, the prevalence
of anti-RHA was compared in groups classified based
on the years between diagnosis and screening test
(Table 2). Two of four patients within a year of diagno-
sis had anti-RHA; however, 9 out of 14 cases of anti-
RHA were after 5 years of diagnosis, suggesting that
anti-RHA does not disappear in Mexican patients with
SLE. This is in striking contrast to the Caucasian popu-
lation [7]. Also, the prevalence of anti-RHA was the
same as or higher than that of anti-Sm in patients even
more than 5 or 10 years after diagnosis. These results
were quite different from the pattern reported in the
previous American study [4]. Distributions of age at
diagnosis, age at anti-RHA test, and years between diag-
nosis and anti-RHA test were compared between anti-
RHA-positive (left in each panel) and -negative (right)
patients (Figure 2a, b, and 2c, respectively). All showed
no clear difference between anti-RHA (+) and (-)
groups.

Levels of anti-RHA antibodies and stage of the disease
and SLEDAI
Next, whether the levels of anti-RHA in patients years
after diagnosis are lower than those of patients with
early-disease status was evaluated by comparing the
intensity of RHA IP and years after diagnosis (Figure 2d).
Although the linear regression analysis suggested a nega-
tive association between these two, it was not statistically
significant. It appeared that the samples with very strong
anti-RHA antibodies and the ones with weak anti-RHA


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Anti-Sm +
Anti-U1RNP +
RHA 1 2 3

U5-200k 3

RHA -o-


p80 >,-
Ku _
n70 I


+ +


4 5 6 7 8 9


1W


PO l-


U 1-ANo- 4W


B'/B p.
Year (Dx-test)


0.1 0.3


4



- 46 .. 12 -
3 4 6 9 12 1212 14 1516 23


b Anti-RHA NHS

RHA1 2 3 4 5 6 7 8 9

r s

Figure 1 Analysis of autoantibodies to RNA helicase A (RHA). (a) Immunoprecipitation using anti-RHA-positive sera. Immunoprecipitation of
31S-methionine-labeled K562 cell extract by anti-RHA-positive sera from Mexican patients with systemic lupus erythematosus (SLE) (n 14), anti-
RHA prototype serum (lane RHA), and a normal human serum (NHS) is shown. Number of years between initial diagnosis and anti-RHA test of
each patient is indicated below the lanes. Positions of RHA, UsnRNP components A, B'/B, U5-200k double, Ku (p70 and p80), Ro 60k, and
ribosomal P PO, and molecular weight (MW) are indicated. Positivity of anti-Sm and U1RNP is indicated at the top. White arrowheads indicate
major degradation products of RHA. (b) Immunoprecipitation and Western blot confirmation of anti-RHA. K562 cell extract was
immunoprecipitated by sera positive for the 140-kDa protein that co-migrated with RHA. Identity of the 140-kDa protein as RHA was validated
by Western blot using a rabbit anti-RHA serum. Lane RHA, anti-RHA prototype serum; lanes 1 to 6, anti-RHA-positive sera screened by
immunoprecipitation; lanes 7 to 9, NHS.


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1314NHS MW
kD
s 205


-116
- 97

- 66




-45





- 29







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Table 1 Frequency of autoantibodies in Mexican patients
with systemic lupus erythematosus
Total Anti-RHA (+) Anti-RHA (-)
Number of patients 62 14 48
RHA 23% (14/62)
U1RNP 29% (18/62) 36% (5/14) 27% (13/48)
Sm 13% (8/62) 21% (3/14) 10% (5/48)
Anti-Sm (+) among 44% (8/18) 60% (3/5) 38% (5/13)
anti-UIRNP (+)
Ribosomal P 8% (5/62) 14% (2/14) 6% (3/48)
Ro 39% (24/62) 29% (4/14) 42% (20/48)
La 8% (5/62) 0% (0/14) 10% (5/48)
Ku 6% (4/62) 14% (2/14) 4% (2/48)


Su
RNAP II
Not significant between ai
Fisher exact test. RHA, RN,


Table 2 Frequency
Years between
diagnosis and test
0 to less than I
1 to less than 2
2 to less than 5
5 to less than 10
10 or more
RHA, RNA helicase A.


were both scattered
disease, suggesting
dramatically, unlike
report [4].
Detailed informa
available from 12
patients. The prev
pared between ant
whether anti-RHA
The prevalence of
lar between grout
RHA (+) SLE had
by comparing SLI
patients, but no cl
No correlation bet
dies and disease ac

Levels of various at
The prevalence of
nificantly differed
-negative patients
-ribosomal P (P pe
by ELISA were co
and -negative case
ence was not appar


The levels of anti-RHA quantified by phosphorimager
and those of anti-UlRNP/Sm by ELISA were compared.
Although the subjects with high levels of anti-RHA may
be more common in the anti-UlRNP/Sm-negative
group, the difference did not reach statistical signifi-
cance (P = 0.07 by Student t test) (Figure 3e). None of
the anti-UlRNP/Sm (+) cases (0/5) had an anti-RHA
integrated density of greater than 30,000 versus 56% (5/
9) in the anti-UlRNP/Sm (-) group (P = 0.086 by Fisher
exact test). There was a trend of negative correlation
between levels of anti-RHA and anti-UIRNP/Sm; how-
ever, it was not statistically significant (Figure 3f).


24% (15/62) 29% (4/14) 23% (11/48) Discussion
10% (6/62) 14% (2/14) 8% (4/48) Anti-Sm antibody is a well-established serological mar-
nti-RHA (+) and (-) groups for all specificities by ker of SLE and is one of the minor criteria under immu-
A helicase A; RNAP II, RNA polymerase II. nological disorders of the SLE classification criteria [3].
However, it is found in only approximately 15% of SLE
of anti-RHA and years from diagnosis patients and in particular it is uncommon in Caucasians
Number of Anti-RHA Anti-Sm [19]. Thus, an additional serological marker specific for
patients (n = 14) (n = 8) SLE should be clinically useful. Anti-RHA has recently
4 50% (2/4) 75% (3/4) been reported as a new serological marker of SLE in the
5 0% (0/5) 0% (0/5) US [4]. The rates of prevalence in the previous study
15 20% (3/15) 0% (0o/15) were 6% in the general SLE population, approximately
17 12% (2/17) 12% (2/17) 6% in Caucasians and African-Americans, and 12% in
21 33% (7/21) 14% (3/21) the Latin population; however, only 3 out of 25 Latin
patients were positive. The present study showed a
much higher prevalence of anti-RHA: 23% in Mexican
patients with SLE. Although anti-RHA appears specific
I throughout the different stages of the for SLE in the previous study regardless of the race, the
that anti-RHA levels do not decrease number of Latin patients was relatively small [4]. Dis-
e in American patients in the previous ease specificity of anti-RHA in other ethnicities, includ-
ing Mexicans, will need to be established in future
tion on SLE classification criteria was studies. In contrast to previous data [4], the presence of
anti-RHA (+) and 42 anti-RHA (-) anti-RHA was not skewed toward patients in an early
alence of each SLE criteria was com- stage of the disease in the current study (Table 1). A
i-RHA (+) and (-) groups to examine comparison of years between diagnosis and anti-RHA
(+) SLE had unique clinical features, test versus levels of anti-RHA (Figure 2d) did not show
all criteria items appeared to be simi- a clear negative correlation, suggesting a relatively stable
ps (data not shown). Whether anti- production of anti-RHA over time in Mexican patients.
different disease activity was evaluated There are several potential explanations for the discre-
EDAI between anti-RHA (+) and (-) pancy. One possibility is the racial difference. The pre-
ear difference was found (Figure 2e). vious study on anti-RHA-positive SLE included 8
:ween the levels of anti-RHA antibo- Caucasians but only 3 African-Americans and 3 Latin
tivity was observed (Figure 2f). patients, indicating that the data were much affected by
the characteristics of Caucasian anti-RHA-positive SLE
itoantibodies and anti-RHA antibodies patients. A recent analysis of the same study population
coexisting autoantibodies was not sig- showed that the majority of Caucasian patients with
nt between anti-RHA-positive and anti-RHA were at an early stage of SLE; 5 out of 8 anti-
(Table 1). Levels of anti-UlRNP/Sm, RHA patients were within one year of diagnosis [7].
ptide), dsDNA, and 132GPI antibodies However, this may not be the case for other races, a cir-
impared between anti-RHA-positive cumstance that may potentially explain the difference.
es (Figure 3a-d); however, the differ- A second difference is a distribution of different stages
rent for any of these specificities. of SLE patients for reasons that are not clear; the


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Age at diagnosis


0
S


.




--l^
0

0,U


(+) ()An
Anti-RHA


B Age at anti-RHA test
80.


60 *


40


20- MO


0
H iI


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Anti-RHA


*

*










(+) (Anti-RHA)
Anti-RHA


80000-
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60000-

40000-

< 20000-

0


SLEDAI


mm

M M


U


000
sees


0*


0 5 10 15 20 25
)iagnosis to anti-RHA test (y)


0 *


(+) (-) 0 40000 80000
Anti-RHA RHA integrated density
Figure 2 Age at diagnosis, age at anti-RNA helicase A (anti-RHA) test, and years between diagnosis and anti-RHA test. Demographic<
data of anti-RHA-positive (n 14) and -negative (n 48) systemic lupus erythematosus (SLE) patients were compared. (a) Age at diagnosis, (b)
Age at anti-RHA test. (c) Years from diagnosis to anti-RHA test. (d) Years from diagnosis to anti-RHA test versus levels of anti-RHA. (e) Systemic
Lupus Erythematosus Disease Activity Index (SLEDAI) in anti-RHA (+) versus (-) patients, (f) Correlation of SLEDAI and levels of anti-RHA. Anti-RH/
levels were measured as integrated density of RHA protein band using phosphorimager as described in Materials and methods, y, years.


Page 6 of 9


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3 40
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Vazquez-Del Mercado et al. Arthritis Research & Therapy 2010, 12:R6
http://arthritis-research.com/content/12/1/R6


Anti-UI RNP/Sm


100,

unit


10



1


(+) (-)
Anti-RHA

Anti-dsDNA


or. "U.="




u* Eu

(+) (-)
Anti-RHA

Anti-RHA levels

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unit

1


F
s 400-

E 300-
U)
S200-

100
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Anti-P peptide


1000
unit
100


10


1


RHA Integrated density


Figure 3 Levels of anti-RNA helicase A (anti-RHA) versus other autoantibodies IgG anti-UI RNP/Sm, ribosomal P (P peptide), double-
stranded DNA (dsDNA), and P2 glycoprotein I (p2GPI) were determined by enzyme-linked immunosorbent assay. Serum dilutions used were
1:2,500 for anti-UI RNP/Sm and 1:500 for all others. Anti-RHA levels were semiquantified from immunoprecipitation using phosphorimager. (a)
Anti-UI RNP/Sm antibodies, (b) Anti-P peptide antibodies, (c) Anti-dsDNA. (d) Anti-p2GPI. (e) Anti-RHA levels in anti-UI RNP/Sm-positive versus
-negative sera. (f) Correlation of anti-RHA versus anti-UI RNP/Sm.


Page 7 of 9


S

m
*U

S*
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Vazquez-Del Mercado et al. Arthritis Research & Therapy 2010, 12:R6
http://arthritis-research.com/content/12/1/R6


previous American study had a large percentage of
patients in an early stage of SLE [4]. In the previous
study, the percentage of patients within 1 or 2 years
into the disease was nearly twice that of the present
study (13% versus 7%, 27% versus 15%, within 1 year
and 2 years, respectively). Although the reduction of
anti-RHA levels did not appear to be a simple result of
immunosuppressive therapy, current and historical treat-
ment may also have effects on the data mentioned
above. These points should be addressed in future stu-
dies with a large number of patients from different
ethnicities.
A hydrocarbon oil pristane induces autoantibodies to
snRNPs, ribosomal P, and Ago2/Su in normal mice
[20,21]; however, it inhibits the production of anti-RHA
antibodies in two strains of mice that spontaneously
produce anti-RHA: NZB/W F1 [12] and CBA/n
(x-linked immunodeficiency) [18]. In the former, pris-
tane induced anti-snRNP and anti-Ago2/Su antibodies
[12]. Human anti-RHA-positive cases that newly devel-
oped anti-snRNPs or -ribosomal P antibodies while their
anti-RHA levels drop significantly [4] appear to be remi-
niscent of the observations in animal models. Thus, it
seems reasonable to hypothesize that the environment
or stage of the disease that is preferable for the produc-
tion of anti-RHA is not ideal for the production of
other specificities. Although the difference was not sta-
tistically significant (P = 0.07, Student t test) (Figure 3e),
five cases with the highest levels of anti-RHA were in
the anti-snRNP-negative group. These may be the cases
that are going to 'switch' the specificities from anti-RHA
to anti-snRNPs later, like the previously described cases
[4]. Factors that are responsible for the production of
anti-RHA or switching from anti-RHA to anti-snRNPs
in humans are not known; however, in animal models of
pristane injection, which switches autoantibody specifi-
city from anti-RHA to anti-snRNP production, type I
interferon (I-IFN) [22,23] and Thl cytokine shifting are
induced [12]. Thus, switching of autoantibody specifici-
ties in humans may also involve changes in cytokine bal-
ance, in particular Thl cytokine or I-IFN production, or
Toll-like receptor (TLR) 7 stimulation since the produc-
tion of pristane-induced anti-snRNPs in animal models
is dependent on I-IFN [24] and TLR7 [25]. Environmen-
tal factors such as viral infection may trigger this type of
change via stimulation of TLRs or via a TLR-indepen-
dent mechanism of I-IFN induction [26]. Although the
production of lupus autoantibodies is generally consid-
ered an event prior to typical clinical manifestation [27],
it is of interest that 10% to 15% of autoantibodies
develop after the diagnosis, in particular within a year of
diagnosis [1]. The development of anti-snRNPs after
steroid treatment reported in clinically MCTD (mixed
connective tissue disease) patients [28,29] is interesting


when considering a role of treatment as a trigger to
change the environment of autoantibody production,
although differentiating the natural course from induc-
tion by steroids is virtually impossible. The identification
of mechanisms of induction and regulation of various
autoantibodies may help develop a strategy of therapeu-
tic regulation of autoantibody production.

Conclusions
The present study reports a high prevalence of anti-
RHA in Mexican patients with SLE. The detection of
SLE-specific autoantibodies in addition to anti-Sm
should be clinically helpful, in particular in the popula-
tion of patients with a low prevalence of anti-Sm.
Patients with high levels of anti-RHA appear to be more
common among anti-snRNP-negative patients. A strong
association of anti-RHA with an early stage of SLE was
not apparent in the Mexican population, possibly due to
the relatively stable production of anti-RHA over time.
Possible differences in clinical significance of anti-RHA
in different races should be clarified in future studies.


Abbreviations
2GPI' 2 glycoprotein 1; ACR' American College of Rheumatology; Ago2'
argonaute 2; dsDNA' double-stranded DNA; ELISA' enzyme-linked
immunosorbent assay; I-IFN' type I interferon; IP' immunoprecipitation; mAb'
monoclonal antibody; OD' optical density; RHA' RNA helicase A; SLE'
systemic lupus erythematosus; SLEDAI' Systemic Lupus Erythematosus
Disease Activity Index; snRNP' small nuclear ribonucleoprotein; TLR' Toll-like
receptor; WB' Western blot

Acknowledgements
This study was supported by CONACyT SEP Ciencia Basica grant 51353
Universidad de Guadalajara agreement 25473 to MVDM MS is supported in
part by a grant from Lupus Foundation of America, Inc EKLC is supported in
part by National Institutes of Health grant A147859

Author details
'Departamento de Biologia Molecular y Gen6mica, Instituto de Investigaci6n
en Reumatologia y del Sistema Musculo Esqueletico, Centro Universitario de
Ciencias de la Salud, Universidad de Guadalajara, Sierra Mojada 950,
Guadalajara, Jalisco, CP 44340, Mexico 2Division de Medicina Interna,
Departamento de Reumatologia, Hospital Civil 'Dr Juan I Menchaca',
Salvador de Quevedo y Zubieta N 750, CP 44340, Guadalajara, Jalisco,
Mexico Departamento de Inmunologia y Reumatologia del Hospital
General de Occidente, Secretara de Salud Jalisco, Av Zoquipan 1050, CP
45100, Zapopan, Jalisco, Mexico 4Division de Investigacin, Hospital de
Especialidades, CMNO, IMSS, and Departamento de Fisiologia, CUCS,
Universidad de Guadalajara, Sierra Mojada 950, CP 44340, Guadalajara, Jalisco,
Mexico 5Departamento de Biologia Molecular y Gen6mica, Instituto de
Biologia Molecular y Gen6mica, Centro Universitario de Ciencias de la Salud,
Universidad de Guadalajara, Sierra Mojada 950, Guadalajara, Jalisco, CP 44340,
Mexico 6Servicio de Medicina-Interna-Reumatologia, Hospital Regional 110,
IMSS, Guadalajara, Jalisco, Mexico 7Division of Rheumatology and Clinical
Immunology, Department of Medicine, University of Florida, 1600 SW Archer
Road, Gainesville, FL 32610-0424, USA 8Department of Oral Biology,
University of Florida, 1600 SW Archer Road, Gainesville, FL 32610-0424, USA
9Department of Pathology, Immunology, and Laboratory Medicine, University
of Florida, 1600 SW Archer Road, Gainesville, FL 32610-0221, USA

Authors' contributions
MVDM and MS designed the study, performed experiments, and wrote the
manuscript CAPS, JFMV, GOB, EOR, RENH, MSP, JAB, JIGN, and LGL helped


Page 8 of 9








Vazquez-Del Mercado et al. Arthritis Research & Therapy 2010, 12:R6
http://arthritis-research.com/content/12/1 /R6


perform experiments and interpret data JYFC and EKLC helped perform
experiments, interpret data, and write the manuscript All authors read and
approved the final manuscript

Competing interests
The authors declare that they have no competing interests

Received: 18 September 2009 Revisions requested: 5 November 2009
Revised: 17 December 2009 Accepted: 8 January 2010
Published: 8 January 2010

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doi:1 0.1186/ar2905
Cite this article as: Vazquez Del Mercado et al High prevalence of
autoantibodies to RNA helicase A in Mexican patients with systemic
lupus erythematosus. Arthritis Research & Therapy 2010 12'R6


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